CLDN5 Mini-Promoters

ABSTRACT

Isolated polynucleotides comprising a CLDN5 mini-promoter are provided. The mini-promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The mini-promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 61/272,466filed Sep. 28, 2009, the disclosure of which application is hereinincorporated by reference.

FIELD OF THE INVENTION

The invention relates to gene promoters and regulatory elements. Morespecifically, the invention relates to CLDN5 promoter compositions andrelated methods.

BACKGROUND

Claudin 5 (CLDN5) a member of the claudin family. Claudins are integralmembrane proteins and components of tight junction strands. Homozygousmutant neonates gradually cease movement and die within 10 hours afterbirth. (Nitta et al. 2003). In the adult mouse brain, CLDN5 is expressedin all major forebrain subdivisions: the neocortex, hippocampus, basalganglia, amygdala/basal forebrain, and olfactory bulb, as well as allother CNS regions (Maynard et al. 2003). CLDN5 is expressed in culturedmouse brain embryonic cells and in freshly isolated MBEC as early asembryonic Day 7. In situ hybridization and immunocytochemical analysesrevealed the presence of the CLDN5 mRNA and its protein product in braincapillary endothelial cells, as well as in a subset of other endothelialand epithelial cells (Chen et al. 1998). In the brain and lung,immunofluorescence microscopy has shown that CLDN5 is exclusivelyconcentrated at cell-cell borders of endothelial cells of all segmentsof blood vessels, but not at those of epithelial cells (Morita et al.1999). Endothelial tight junctions are an important functional part ofthe formation of the blood brain barrier, and CLDN5 has been shown to bea determinant of blood brain barrier permeability (Matter and Balda2003; Nitta et al. 2003; Stamatovic et al. 2008).

Functional mouse CLDN5 promoter sequences have been identified andanalyzed (Burek and Forster 2009). In this paper, the authors performluciferase reporter assays in a brain microvascular endothelial cellline, but do not report any in vivo expression data. A 1.5 kb humanCLDN5 promoter sequence was tested for expression in bovine retinalendothelial cells (Felinski et al. 2008).

Minimal human promoter elements which are capable of driving expressionin specific cell types and/or in specific regions of the brain are ofinterest. Also of interest is the identification of minimal elementsrequired for adequate expression and specificity that allow ease of usein expression constructs.

RELEVANT LITERATURE

References of interest include:

Burek, M. and C. Y. Forster (2009). “Cloning and characterization of themurine claudin-5 promoter.” Mol Cell Endocrinol 298(1-2): 19-24;

Chen, Z., M. Zandonatti, et al. (1998). “Brain capillary endothelialcells express MBEC1, a protein that is related to the Clostridiumperfringens enterotoxin receptors.” Lab Invest 78(3): 353-63;

Felinski, E. A., A. E. Cox, et al. (2008). “Glucocorticoids inducetransactivation of tight junction genes occludin and claudin-5 inretinal endothelial cells via a novel cis-element.” Exp Eye Res 86(6):867-78;

Jasin, M., M. E. Moynahan, et al. (1996) “Targeted transgenesis.” ProcNatl Acad Sci USA 93(17): 8804-8;

Matter, K. and M. S. Balda (2003). “Holey barrier: claudins and theregulation of brain endothelial permeability.” J Cell Biol 161(3):459-60;

Maynard, T. M., G. T. Haskell, et al. (2003). “A comprehensive analysisof 22q11 gene expression in the developing and adult brain.” Proc NatlAcad Sci USA 100(24): 14433-8;

Morita, K., H. Sasaki, et al. (1999). “Endothelial claudin:claudin-5/TMVCF constitutes tight junction strands in endothelialcells.” J Cell Biol 147(1): 185-94;

Nitta, T., M. Hata, et al. (2003). “Size-selective loosening of theblood-brain barrier in claudin-5-deficient mice.” J Cell Biol 161(3):653-60; and

Stamatovic, S. M., R. F. Keep, et al. (2008). “Brain endothelialcell-cell junctions: how to “open” the blood brain barrier.” CurrNeuropharmacol 6(3): 179-92.

SUMMARY OF THE INVENTION

Provided herein are nucleic acid compositions and methods relating toCLDN5 promoters having a sequence other than a native CLDN5 promoter.

In one embodiment of the invention, there is provided an isolatednucleic acid fragment comprising a CLDN5 mini-promoter, wherein theCLDN5 mini-promoter comprises one or more CLDN5 regulatory elementsoperably linked in a non-native conformation to a CLDN5 basal promoter.In other embodiments, there is provided an isolated nucleic acidfragment comprising a CLDN5 mini-promoter, wherein the CLDN5mini-promoter comprises a CLDN5 basal promoter. The CLDN5 mini-promotermay have a nucleic acid sequence that is substantially similar insequence and function to SEQ ID NO: 1 or 2. The one or more CLDN5regulatory elements may have nucleic acid sequences that aresubstantially similar in sequence and function to SEQ ID NO: 3, SEQ IDNO: 4, and/or SEQ ID NO: 5. The CLDN5 basal promoter may have a nucleicacid sequence which is substantially similar in sequence and function toSEQ ID NO: 6. The CLDN5 mini-promoter may further be operably linked toan expressible sequence, e.g. reporter genes, genes encoding apolypeptide of interest, regulatory RNA sequences such as miRNA, siRNA,anti-sense RNA, etc., and the like. Reporter gene sequences include, forexample luciferase, beta-galactosidase, green fluorescent protein,enhanced green fluorescent protein, and the like as known in the art.The expressible sequence may encode a protein of interest, for example atherapeutic protein, receptor, antibody, growth factor, and the like.The expressible sequence may encode an RNA interference or antisensemolecule.

In one embodiment, there is provided an expression vector comprising aCLDN5 mini-promoter element, wherein the CLDN5 mini-promoter comprisesone or more CLDN5 regulatory elements operably linked in a non-nativeconformation to a CLDN5 basal promoter. In other embodiments, there isprovided an expression vector comprising a CLDN5 mini-promoter, whereinthe CLDN5 mini-promoter comprises a CLDN5 basal promoter. The CLDN5mini-promoter may have a nucleic acid sequence which is substantiallysimilar in sequence and function to SEQ ID NO: 1 or 2. The one or moreCLDN5 regulatory elements may have nucleic acid sequences which aresubstantially similar in sequence and function to SEQ ID NO: 3, SEQ IDNO: 4, and/or SEQ ID NO: 5. The CLDN5 basal promoter may have a nucleicacid sequence which is substantially similar in sequence and function toSEQ ID NO: 6. The CLDN5 mini-promoter may further be operably linked toan expressible sequence, e.g. reporter genes, genes encoding apolypeptide of interest, regulatory RNA sequences such as miRNA, siRNA,anti-sense RNA, etc., and the like. Reporter gene sequences include, forexample luciferase, beta-galactosidase, green fluorescent protein,enhanced green fluorescent protein, and the like as known in the art.The expressible sequence may encode a protein of interest, for example atherapeutic protein, receptor, antibody, growth factor, and the like.The expressible sequence may encode an RNA interference molecule. Theexpression vector may further comprise a genomic targeting sequence. Thegenomic targeting sequence may be HPRT.

In one embodiment, there is provided a method for expressing a gene,protein, RNA interference molecule or the like in a cell, the methodcomprising introducing into the cell a expression vector comprising aCLDN5 mini-promoter element, wherein the CLDN5 mini-promoter comprisesone or more CLDN5 regulatory elements operably linked in a non-nativeconformation to a CLDN5 basal promoter. In other embodiments, there isprovided a method for expressing a gene, protein, RNA interferencemolecule or the like in a cell, the method comprising introducing intothe cell an expression vector comprising a CLDN5 mini-promoter, whereinthe CLDN5 mini-promoter comprises a CLDN5 basal promoter. The CLDN5mini-promoter may have a nucleic acid sequence which is substantiallysimilar in sequence and function to SEQ ID NO: 1 or 2. The one or moreCLDN5 regulatory elements may have nucleic acid sequences which aresubstantially similar in sequence and function to SEQ ID NO: 3, SEQ IDNO: 4, and/or SEQ ID NO: 5. The CLDN5 basal promoter may have a nucleicacid sequence which is substantially similar in sequence and function toSEQ ID NO: 6. Cells of interest may include blood vessel cells in thebrain. The CLDN5 mini-promoter may further be operably linked to anexpressible sequence, e.g. reporter genes, genes encoding a polypeptideof interest, regulatory RNA sequences such as miRNA, siRNA, anti-senseRNA, etc., and the like. Reporter gene sequences include, for exampleluciferase, beta-galactosidase, green fluorescent protein, enhancedgreen fluorescent protein, and the like as known in the art. Theexpressible sequence may encode a protein of interest, for example atherapeutic protein, receptor, antibody, growth factor, and the like.The expressible sequence may encode an RNA interference molecule, or anantisense RNA molecule. The expression vector may thus further comprisea genomic targeting sequence. The genomic targeting sequence may beHPRT.

In one embodiment of the invention, there is provided a method foridentifying or labeling a cell, the method comprising introducing intothe cell a expression vector comprising a CLDN5 mini-promoter elementoperably linked to an expressible sequence, wherein the CLDN5mini-promoter element comprises one or more CLDN5 regulatory elementsoperably linked in a non-native conformation to a CLDN5 basal promoterelement, and wherein the expressible sequence comprises a reporter gene.In another embodiment, the CLDN5 mini-promoter element comprises a CLDN5basal promoter element. The CLDN5 mini-promoter may have a nucleic acidsequence which is substantially similar in sequence and function to SEQID NO: 1 or 2. The one or more CLDN5 regulatory elements may havenucleic acid sequences which are substantially similar in sequence andfunction to SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 5. The CLDN5basal promoter may have a nucleic acid sequence which is substantiallysimilar in sequence and function to SEQ ID NO: 6. In some embodiments,the cell is a blood vessel cell in the brain. Reporter gene sequencesinclude, for example luciferase, beta-galactosidase, green fluorescentprotein, enhanced green fluorescent protein, and the like as known inthe art. The expressible sequence may encode a protein of interest, forexample a therapeutic protein, receptor, antibody, growth factor, RNAinterference molecule and the like.

In one embodiment of the invention, there is provided a method formonitoring or tracking the development or maturation of a cell, themethod comprising: 1) introducing into the cell a expression vectorcomprising a CLDN5 mini-promoter element operably linked to anexpressible sequence, wherein the CLDN5 mini-promoter element comprisesone or more CLDN5 regulatory elements operably linked in a non-nativeconformation to a CLDN5 basal promoter element, and wherein theexpressible sequence comprises a reporter gene; and 2) detecting theexpression of the reporter gene in the progeny of the cell as a means ofdetermining the lineage, identity or developmental state of theprogenitor cell or progeny thereof. In other embodiments, the CLDN5mini-promoter comprises a CLDN5 basal promoter. The CLDN5 mini-promotermay have a nucleic acid sequence which is substantially similar insequence and function to SEQ ID NO: 1 or 2. The one or more CLDN5regulatory elements may have nucleic acid sequences which aresubstantially similar in sequence and function to SEQ ID NO: 3, SEQ IDNO: 4, and/or SEQ ID NO: 5. The CLDN5 basal promoter may have a nucleicacid sequence which is substantially similar in sequence and function toSEQ ID NO: 6. In some embodiments, the cell may be a blood vessel cellof the brain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Vector diagram of backbone pEMS1313

FIG. 2: Expression of reporter by CLDN5 promoter elements in bloodvessels in brain (Ple32)

FIG. 3: Expression of reporter by CLDN5 promoter elements in bloodvessels in brain (Ple34)

FIG. 4: Expression of reporter by CLDN5 promoter elements in spinal cord(Ple34)

DETAILED DESCRIPTION

Compositions that include novel polynucleotides comprising CLDN5promoter elements (also referred to herein as CLDN5 mini-promoters) aswell as novel expression vectors comprising said CLDN5 promoter elements(or mini-promoters), are provided. Also provided are various methodsutilizing the subject CLDN5 promoter (or mini-promoter) elements orexpression vectors.

The term ‘CLDN5’ refers to the gene which encodes the CLDN5 protein,also referred to as AWAL, BEC1, CPETRL1, TMVCF, and also as MBEC1 inmouse. The human homolog of CLDN5 is encoded by the human geneidentified as EntrezGene #7122, and is located at chromosomal location22q11.21. The protein encoded by human CLDN5 has the Protein Accession#000501.1 and/or Q53HW4 (Swiss-Prot). Other mammalian CLDN5 homologsinclude but are not limited to: Rattus norvegicus (EntrezGene #65131,Protein Accession #Q9JKD6.2), Mus musculus (EntrezGene #12741, ProteinAccession #O54942.2).

The term ‘promoter’ refers to the regulatory DNA region which controlstranscription or expression of a gene and which can be located adjacentto or overlapping a nucleotide or region of nucleotides at which RNAtranscription is initiated. A promoter contains specific DNA sequenceswhich bind protein factors, often referred to as transcription factors,which facilitate binding of RNA polymerase to the DNA leading to genetranscription. A ‘basal promoter’, also referred to as a ‘corepromoter’, usually means a promoter which contains all the basicnecessary elements to promote transcriptional expression of an operablylinked polynucleotide. An ‘CLDN5 basal promoter’, in the context of thepresent invention and as used herein, is a nucleic acid compound havinga sequence with at least 65%, at least 70%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 99% similarity to SEQ ID NO: 6,and which comprises at least 1, usually at least 2, and most usually atleast 4 of the identified conserved sequences listed in Table 1.

TABLE 1 List of conserved sequences in the human CLDN5 basal promoter -SEQ ID NO: 6. The start and end coordinates of the sequences arerelative to the full SEQ ID NO: 6 sequence. Start (relative to End(relative to Ple32 sequence) Ple32 sequence) Invariant sequence type 346522 Conserved sequence and validated SOX18 binding site 1400 1405Conserved sequence 1430 1436 Conserved sequence 1474 1479 Conservedsequence

A promoter may also include one or more ‘regulatory elements’ which mayalso influence the expression or transcription by the promoter. Suchregulatory elements encode specific DNA sequences which bind otherfactors, which may include but are not limited to enhancers, silencers,insulators, and/or boundary elements. An ‘CLDN5 regulatory element’, inthe context of the present invention and as used herein, may be anucleic acid compound having a sequence with at least 65%, at least 70%,at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%similarity to SEQ ID NO: 3, 4, or 5. The present invention provides, incertain embodiments as described herein, different promoters of theCLDN5 gene. In some embodiments, the CLDN5 promoter comprises one ormore CLDN5 regulatory elements operably linked to a CLDN5 basalpromoter. In certain embodiments, the CLDN5 regulatory elements aredirectly joined with no intervening sequences. In other embodiments, theCLDN5 regulatory elements may be operably linked with interveningsequences. In general the spacing between the regulatory elements is notmore than about 15 KB, generally not more than about 10 KB, usually notmore than about 1 KB, more often not more than about 500 nt, and may benot more than about 100 nt, down to a direct joining of the twosequences.

The term ‘operably linked’, in the context of the present invention,means joined in such a fashion as to work together to allowtranscription. In some embodiments of the invention, two polynucleotidesequences may be operably linked by being directly linked via anucleotide bond. In this fashion, the two operably linked elementscontain no intervening sequences and in being joined are able to directtranscription of an expression sequence. In other embodiments of theinvention, two elements may be operably linked by an interveningcompound, for instance a polynucleotide sequence of variable length. Insuch a fashion, the operably linked elements, although not directlyjuxtaposed, are still able to direct transcription of an expressionsequence. Thus, according to some embodiments of the invention, one ormore promoter elements may be operably linked to each other, andadditionally be operably linked to a downstream expression sequence,such that the linked promoter elements are able to direct expression ofthe downstream expression sequence.

The term ‘mini-promoter’ refers to a promoter in which certain promoterelements are combined in a non-native conformation, usually in such afashion as to reduce the overall size of the promoter compared to thenative conformation. For example, after identification of criticalpromoter elements, using one or more of various techniques, the nativesequences that intervene between the identified elements may bepartially or completely removed. Other non-native sequences mayoptionally be inserted between the identified promoter elements. Theterm mini-promoter may also refer to a minimal promoter element in anative conformation which is capable of driving protein expression, butwhich has had non-essential elements removed in order to reduce itssize. A mini-promoter may provide certain advantages over largerpromoter conformations. For example, the smaller size of themini-promoter may allow easier genetic manipulation, for example in thedesign and/or construction of expression vectors or other recombinantDNA constructs. In addition, the smaller size may allow easier insertionof DNA constructs into host cells and/or genomes, for example viatransfection, transformation, etc. Other advantages of mini-promoterswould be apparent to one of skill in the art. In some embodiments of theinvention, there are thus provided novel CLDN5 mini-promoters comprisingone or more CLDN5 regulatory elements operably linked in a non-nativeconformation to a CLDN5 basal promoter. In general the spacing betweenthe one or more CLDN5 regulatory elements and the CLDN5 basal promoteris not more than about 15 KB, generally not more than about 10 KB,usually not more than about 1 KB, more often not more than about 500 nt,and may be not more than about 100 nt, down to a direct joining of thetwo sequences. In other embodiments, there are provided novel CLDN5mini-promoters comprising a CLDN5 basal promoter.

The term ‘expressible sequence’ refers to a polynucleotide compositionwhich is operably linked to a promoter element such that the promoterelement is able to cause transcriptional expression of the expressionsequence. An expressible sequence is typically linked downstream, on the3′-end of the promoter element(s) in order to achieve transcriptionalexpression. The result of this transcriptional expression is theproduction of an RNA macromolecule. The expressed RNA molecule mayencode a protein and may thus be subsequently translated by theappropriate cellular machinery to produce a polypeptide proteinmolecule. In some embodiments of the invention, the expression sequencemay encode a reporter protein. Alternately, the RNA molecule may be anantisense, RNAi or other non-coding RNA molecule, which may be capableof modulating the expression of specific genes in a cell, as is known inthe art.

The term ‘RNA’ as used in the present invention includes full-length RNAmolecules, which may be coding or non-coding sequences, fragments, andderivatives thereof. For example, a full-length RNA may initiallyencompass up to about 20 Kb or more of sequence, and frequently will beprocessed by splicing to generate a small mature RNA. Fragments, RNAi,miRNA and anti-sense molecules may be smaller, usually at least about 18nt. in length, at least about 20 nt in length, at least about 25 nt. inlength, and may be up to about 50 nt. in length, up to about 100 nt inlength, or more. RNA may be single stranded, double stranded, synthetic,isolated, partially isolated, essentially pure or recombinant. RNAcompounds may be naturally occurring, or they may be altered such thatthey differ from naturally occurring RNA compounds. Alterations mayinclude addition, deletion, substitution or modification of existingnucleotides. Such nucleotides may be either naturally occurring, ornon-naturally occurring nucleotides. Alterations may also involveaddition or insertion of non-nucleotide material, for instance at theend or ends of an existing RNA compound, or at a site that is internalto the RNA (ie. between two or more nucleotides).

The term ‘nucleic acid’ as used herein includes any nucleic acid, andmay be a deoxyribonucleotide or ribonucleotide polymer in either singleor double-stranded form. A ‘polynucleotide’ or ‘nucleotide polymer’ asused herein may include synthetic or mixed polymers of nucleic acids,both sense and antisense strands, and may be chemically or biochemicallymodified or may contain non- natural or derivatized nucleotide bases, aswill be readily appreciated by those skilled in the art. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.),charged linkages (e. g., phosphorothioates, phosphorodithioates, etc.),pendent moieties (e.g., polypeptides), and modified linkages (e.g.,alpha anomeric polynucleotides, etc.). Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions.

A ‘purine’ is a heterocyclic organic compound containing fusedpyrimidine and imidazole rings, and acts as the parent compound forpurine bases, adenine (A) and guanine (G). ‘Nucleotides’ are generally apurine (R) or pyrimidine (Y) base covalently linked to a pentose,usually ribose or deoxyribose, where the sugar carries one or morephosphate groups. Nucleic acids are generally a polymer of nucleotidesjoined by 3′ 5′ phosphodiester linkages. As used herein ‘purine’ is usedto refer to the purine bases, A and G, and more broadly to include thenucleotide monomers, deoxyadenosine-5′ -phosphate anddeoxyguanosine-5′-phosphate, as components of a polynucleotide chain. A‘pyrimidine’ is a single-ringed, organic base that forms nucleotidebases, such as cytosine (C), thymine (T) and uracil (U). As used herein‘pyrimidine’ is used to refer to the pyrimidine bases, C, T and U, andmore broadly to include the pyrimidine nucleotide monomers that alongwith purine nucleotides are the components of a polynucleotide chain.

It is within the capability of one of skill in the art to modify thesequence of a promoter nucleic acid sequence, e.g. the provided basalpromoter and/or regulatory sequences, in a manner that does notsubstantially change the activity of the promoter element, i.e. thetranscription rate of an expressible sequence operably linked to amodified promoter sequence is at least about 65% the transcription rateof the original promoter, at least about 75% the transcription rate ofthe original promoter sequence, at least about 80%, at least about 90%,at least about 95%, at least about 99%, or more. Such modified sequenceswould be considered to be ‘functionally similar’ or to have ‘functionalsimilarity’ or ‘substantial functional similarity’ to the unmodifiedsequence. Such modifications may include insertions, deletions which maybe truncation of the sequence or internal deletions, or substitutions.The level of sequence modification to an original sequence willdetermine the ‘sequence similarity’ of the original and modifiedsequences. Modification of the promoter elements of the presentinvention in a fashion that does not significantly alter transcriptionalactivity, as described above would result in sequences with ‘substantialsequence similarity’ to the original sequence i.e. the modified sequencehas a nucleic acid composition that is at least about 65% similar to theoriginal promoter sequence, at least about 75% similar to the originalpromoter sequence, at least about 80%, at least about 90%, at leastabout 95%, at least about 99%, or more similar to the original promotersequence. Thus, mini-promoter elements which have substantial functionaland/or sequence similarity are herein described and are within the scopeof the invention.

An ‘RNA interference molecule’, or ‘RNA interference sequence’ asdefined herein, may include, but is not limited to, an antisense RNAmolecule, a microRNA molecule or a short hairpin RNA (shRNA) molecule.Typically, RNA interference molecules are capable of target-specificmodulation of gene expression and exert their effect either by mediatingdegradation of the mRNA products of the target gene, or by preventingprotein translation from the mRNA of the target gene. The overall effectof interference with mRNA function is modulation of expression of theproduct of a target gene. This modulation can be measured in ways whichare routine in the art, for example by Northern blot assay or reversetranscriptase PCR of mRNA expression, Western blot or ELISA assay ofprotein expression, immunoprecipitation assay of protein expression,etc.

An ‘antisense RNA molecule’, as used herein, is typically a singlestranded RNA compound which binds to complementary RNA compounds, suchas target mRNA molecules, and blocks translation from the complementaryRNA compounds by sterically interfering with the normal translationalmachinery. Specific targeting of antisense RNA compounds to inhibit theexpression of a desired gene may design the antisense RNA compound tohave a homologous, complementary sequence to the desired gene. Perfecthomology is not necessary for inhibition of expression. Design of genespecific antisense RNA compounds, including nucleotide sequenceselection and additionally appropriate alterations, are known to one ofskill in the art.

The term ‘rnicroRNA molecule’, ThicroRNA' or ThiRNA', as used herein,refers to single-stranded RNA molecules, typically of about 21-23nucleotides in length, which are capable of modulating gene expression.Mature miRNA molecules are partially complementary to one or moremessenger RNA (mRNA) molecules, and their main function is todownregulate gene expression. Without being bound by theory, miRNAs arefirst transcribed as primary transcripts or pri-miRNA with a cap andpoly-A tail and processed to short, 70-nucleotide stem-loop structuresknown as pre-miRNA in the cell nucleus. This processing is performed inanimals by a protein complex known as the Microprocessor complex,consisting of the nuclease Drosha and the double-stranded RNA bindingprotein Pasha. These pre-miRNAs are then processed to mature miRNAs inthe cytoplasm by interaction with the endonuclease Dicer, which alsoinitiates the formation of the RNA-induced silencing complex (RISC).When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNAmolecules are formed, but only one is integrated into the RISC complex.This strand is known as the guide strand and is selected by theargonaute protein, the catalytically active RNase in the RISC complex,on the basis of the stability of the 5′ end. The remaining strand, knownas the anti-guide or passenger strand, is degraded as a RISC complexsubstrate. After integration into the active RISC complex, miRNAs basepair with their complementary mRNA molecules and induce mRNA degradationby argonaute proteins, the catalytically active members of the RISCcomplex. Animal miRNAs are usually complementary to a site in the 3′ UTRwhereas plant miRNAs are usually complementary to coding regions ofmRNAs.

The term ‘short hairpin RNA’ or ‘shRNA’ refers to RNA molecules havingan RNA sequence that makes a tight hairpin turn that can be used tosilence gene expression via RNA interference. The shRNA hairpinstructure is cleaved by the cellular machinery into siRNA, which is thenbound to the RNA-induced silencing complex (RISC). This complex binds toand cleaves mRNAs which match the siRNA that is bound to it. shRNA istranscribed by RNA Polymerase III whereas miRNA is transcribed by RNAPolymerase II. Techniques for designing target specific shRNA moleculesare known in the art.

An ‘expression vector’ is typically a nucleic acid molecule which is maybe integrating or autonomous, (i.e. self-replicating), and whichcontains the necessary components to achieve transcription of anexpressible sequence in a target cell, when introduced into the targetcell. Expression vectors may include plasmids, cosmids, phage, YAC, BAC,mini-chromosomes, viruses, e.g. retroviruses, adenovirus, lentivirus,SV-40, and the like; etc. Many such vectors have been described in theart and are suitable for use with the promoters of the presentinvention. Expression vectors of the present invention include apromoter as described herein, operably linked to an expressiblesequence, which may also be optionally operably linked to atranscription termination sequence, such as a polyadenylation sequence.The expression vector optionally contains nucleic acid elements whichconfer host selectivity, elements that facilitate replication of thevector, elements that facilitate integration of the vector into thegenome of the target cell, elements which confer properties, for exampleantibiotic resistance, to the target cell which allow selection orscreening of transformed cells and the like. Techniques and methods fordesign and construction of expression vectors are well known in the art.

It may be desirable, when driving expression of an expressible sequencewith a particular promoter system to have the expression occur in astable and consistent manner. A factor that has been shown to affectexpression is the site of integration of an expression vector orconstruct into the genome of the target cell, sometimes called ‘positioneffects’. Such position effects may be caused by, for example, localchromatin structure which affects expression of sequences from thatregion of the genome. One method to control for position effects whenintegrating an expression vector or construct into the genome of atarget cell is to include a ‘genomic targeting sequence’ in the vectoror construct that directs integration of the vector or construct to aspecific genomic site. As an example, the hypoxanthinephosphoribosyltransferase (HPRT) gene has been used successfully forthis purpose (Bronson et al. 1996; Jasin et al. 1996). The HPRT gene hasadditional advantages as a genomic targeting sequence, for instance itsconcomitant use as a selectable marker system. Other genomic targetingsequences that may be useful in the present invention are described inthe art, for instance (Jasin et al. 1996; van der Weyden et al. 2002).The genomic targeting signals as described herein are useful in certainembodiments of the present invention.

Introduction of nucleic acids or expression vectors may be accomplishedusing techniques well known in the art, for example microinjection,electroporation, particle bombardment, or chemical transformation, suchas calcium-mediated transformation, as described for example in Maniatiset al. 1982, Molecular Cloning, A laboratory Manual, Cold Spring HarborLaboratory or in Ausubel et al. 1994, Current protocols in molecularbiology, JoIm Wiley and Sons.

CLDN5 Promoters

The present invention herein provides novel CLDN5 mini-promotersequences which are capable of effecting transcriptional expression in aspatial and temporal fashion which may be similar to naturally occurringCLDN5 promoters, although the invention may also provide for variationaway from the native expression patterns. In some embodiments, the CLDN5mini-promoters of the invention comprise CLDN5 promoter elements joinedin a non-native configuration, thus providing advantageouscharacteristics. In other embodiments, the CLDN5 mini-promoters comprisebasal promoters with advantageous characteristics. Also provided arenovel expression vector compositions comprising CLDN5 mini-promoterswhich allow consistent specific spatiotemporal transcription ofexpression sequences. Also provided are novel methods utilizing theseCLDN5 mini-promoters and expression vectors.

The CLDN5 mini-promoters of some embodiments of the invention, asdescribed herein, comprise CLDN5 promoter elements that are joined bynon-native sequences. In this context, the native intervening sequencesmay have been partially or completely removed, and optionally may havebeen replaced with non-native sequences. In such a fashion, the naturalspacing of the promoter elements, for instance the human CLDN5regulatory elements corresponding to SEQ ID NO: 3, SEQ ID NO: 4 and/orSEQ ID NO: 5 and the human CLDN5 basal promoter element corresponding toSEQ ID NO: 6, or sequences with substantial functional and/or sequenceequivalence, is altered. An advantage of such non-native mini-promotersis that the removal of native intervening sequences reduces the size ofthe mini-promoter while maintaining the functional activity of thepromoter, thus improving the utility of the mini-promoter for variousapplications. The CLDN5 mini-promoters of other embodiments of theinvention comprise CLDN5 basal promoters. The advantage of such basalpromoters are also related to the significantly reduced size as comparedto native promoters.

In certain embodiments, human CLDN5 mini-promoters having a sequencescorresponding to SEQ ID NO: 1 and 2 direct expression of an expressiblesequence which is operably linked downstream of the CLDN5 promoter inspecific cell types in different regions of the brain (see e.g., thenon-limiting working examples). The CLDN5 regulatory elements (SEQ IDNOs: 3, 4, 5) and CLDN5 basal promoter element (SEQ ID NO: 6) havesequences which are identical to those found upstream of the human CLDN5gene.

Promoters of the present invention may be modified with respect to thenative regulatory and/or native basal promoter sequence. In general,such modifications will not change the functional activity of thepromoter with respect to cell-type selectivity; and to the rate oftranscription in cells where the promoter is active. The modifiedmini-promoter provide for a transcription rate of an expressiblesequence operably linked to a modified promoter sequence that is atleast about 75% the transcription rate of the promoter sequence of SEQID NO:1 or 2, at least about 80%, at least about 90%, at least about95%, at least about 99%, or more. Methods of assessing promoter strengthand selectivity are known in the art, including, for example, expressionof a reporter sequence in a cell in vivo or in vitro, and quantitatingthe reporter activity.

In one embodiment of the invention, there is provided an isolatednucleic acid fragment comprising a CLDN5 mini-promoter, wherein theCLDN5 mini-promoter comprises one or more CLDN5 regulatory elementsoperably linked in a non-native conformation to a CLDN5 basal promoter.In other embodiments, there is provided an isolated nucleic acidfragment comprising a CLDN5 mini-promoter, wherein the CLDN5mini-promoter comprises a CLDN5 basal promoter. The CLDN5 mini-promotermay have a nucleic acid sequence which is substantially similar insequence and function to SEQ ID NO: 1 or 2. The one or more CLDN5regulatory elements may have nucleic acid sequences which aresubstantially similar in sequence and function to SEQ ID NO: 3, SEQ IDNO: 4, and/or SEQ ID NO: 5. The CLDN5 basal promoter may have a nucleicacid sequence which is substantially similar in sequence and function toSEQ ID NO: 6. The CLDN5 mini-promoter may further be operably linked toan expressible sequence, e.g. reporter genes, genes encoding apolypeptide of interest, regulatory RNA sequences such as miRNA, siRNA,anti-sense RNA, etc., and the like. Reporter gene sequences include, forexample luciferase, beta-galactosidase, green fluorescent protein,enhanced green fluorescent protein, and the like as known in the art.The expressible sequence may encode a protein of interest, for example atherapeutic protein, receptor, antibody, growth factor, and the like.The expressible sequence may encode an RNA interference molecule.

Means of expressing a gene, protein, RNA interference molecule or thelike in a cell, tissue or organ, are provided, utilizing the subjectexpression vectors comprising CLDN5 mini-promoters. In one embodiment,there is provided an expression vector comprising a CLDN5 mini-promoterelement, wherein the CLDN5 mini-promoter comprises one or more CLDN5regulatory elements operably linked in a non-native conformation to aCLDN5 basal promoter. In other embodiments, there is provided anexpression vector comprising a CLDN5 mini-promoter, wherein the CLDN5mini-promoter comprises a CLDN5 basal promoter. The CLDN5 mini-promotermay have a nucleic acid sequence which is substantially similar insequence and function to SEQ ID NO: 1 or 2. The one or more CLDN5regulatory elements may have nucleic acid sequences which aresubstantially similar in sequence and function to SEQ ID NO: 3, SEQ IDNO: 4, and/or SEQ ID NO: 5. The CLDN5 basal promoter may have a nucleicacid sequence which is substantially similar in sequence and function toSEQ ID NO: 6. The CLDN5 mini-promoter may further be operably linked toan expressible sequence, e.g. reporter genes, genes encoding apolypeptide of interest, regulatory RNA sequences such as miRNA, siRNA,anti-sense RNA, etc., and the like. Reporter gene sequences include, forexample luciferase, beta-galactosidase, green fluorescent protein,enhanced green fluorescent protein, and the like as known in the art.The expressible sequence may encode a protein of interest, for example atherapeutic protein, receptor, antibody, growth factor, and the like.The expressible sequence may encode an RNA interference molecule. Theexpression vector may further comprise a genomic targeting sequence. Thegenomic targeting sequence may be HPRT.

In certain embodiments, the subject expression vectors comprising novelCLDN5 mini-promoter elements direct transcription of an expressionsequence in specific cell types in specific regions of the brain andbody, most notably the blood vessels in the brain. In some embodimentsof the invention, there is thus provided a method for expressing a gene,protein, RNA interference molecule or the like in the targeted cells ofthe brain. In one embodiment, there is provided a method for expressinga gene, protein, RNA interference molecule or the like in a cell, themethod comprising introducing into the cell a expression vectorcomprising a CLDN5 mini-promoter element, wherein the CLDN5mini-promoter comprises one or more CLDN5 regulatory elements operablylinked in a non-native conformation to a CLDN5 basal promoter. In otherembodiments, there is provided a method for expressing a gene, protein,RNA interference molecule or the like in a cell, the method comprisingintroducing into the cell an expression vector comprising a CLDN5mini-promoter, wherein the CLDN5 mini-promoter comprises a CLDN5 basalpromoter. The CLDN5 mini-promoter may have a nucleic acid sequence whichis substantially similar in sequence and function to SEQ ID NO: 1 or 2.The one or more CLDN5 regulatory elements may have nucleic acidsequences which are substantially similar in sequence and function toSEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 5. The CLDN5 basalpromoter may have a nucleic acid sequence which is substantially similarin sequence and function to SEQ ID NO: 6. Cells of interest may includeblood vessel cells in the brain. The CLDN5 mini-promoter may further beoperably linked to an expressible sequence, e.g. reporter genes, genesencoding a polypeptide of interest, regulatory RNA sequences such asmiRNA, siRNA, anti-sense RNA, etc., and the like. Reporter genesequences include, for example luciferase, beta-galactosidase, greenfluorescent protein, enhanced green fluorescent protein, and the like asknown in the art. The expressible sequence may encode a protein ofinterest, for example a therapeutic protein, receptor, antibody, growthfactor, and the like. The expressible sequence may encode an RNAinterference molecule. The expression vector may thus further comprise agenomic targeting sequence. The genomic targeting sequence may be HPRT.

In one embodiment of the invention, there is provided a method foridentifying or labeling a cell, the method comprising introducing intothe cell a expression vector comprising a CLDN5 mini-promoter elementoperably linked to an expressible sequence, wherein the CLDN5mini-promoter element comprises one or more CLDN5 regulatory elementsoperably linked in a non-native conformation to a CLDN5 basal promoterelement, and wherein the expressible sequence comprises a reporter gene.In another embodiment, the CLDN5 mini-promoter element comprises a CLDN5basal promoter element. The CLDN5 mini-promoter may have a nucleic acidsequence which is substantially similar in sequence and function to SEQID NO: 1 or 2. The one or more CLDN5 regulatory elements may havenucleic acid sequences which are substantially similar in sequence andfunction to SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 5. The CLDN5basal promoter may have a nucleic acid sequence which is substantiallysimilar in sequence and function to SEQ ID NO: 6. In some embodiments,the cell is a blood vessel cell in the brain. Reporter gene sequencesinclude, for example luciferase, beta-galactosidase, green fluorescentprotein, enhanced green fluorescent protein, and the like as known inthe art. The expressible sequence may encode a protein of interest, forexample a therapeutic protein, receptor, antibody, growth factor, RNAinterference molecule and the like.

In one embodiment of the invention, there is provided a method formonitoring or tracking the development or maturation of a cell, themethod comprising: 1) introducing into the cell a expression vectorcomprising a CLDN5 mini-promoter element operably linked to anexpressible sequence, wherein the CLDN5 mini-promoter element comprisesone or more CLDN5 regulatory elements operably linked in a non-nativeconformation to a CLDN5 basal promoter element, and wherein theexpressible sequence comprises a reporter gene; and 2) detecting theexpression of the reporter gene in the progeny of the cell as a means ofdetermining the lineage, identity or developmental state of theprogenitor cell or progeny thereof. In other embodiments, the CLDN5mini-promoter comprises a CLDN5 basal promoter. The CLDN5 mini-promotermay have a nucleic acid sequence which is substantially similar insequence and function to SEQ ID NO: 1 or 2. The one or more CLDN5regulatory elements may have nucleic acid sequences which aresubstantially similar in sequence and function to SEQ ID NO: 3, SEQ IDNO: 4, and/or SEQ ID NO: 5. The CLDN5 basal promoter may have a nucleicacid sequence which is substantially similar in sequence and function toSEQ ID NO: 6. In some embodiments, the cell may be a blood vessel cellof the brain.

The inventors herein further describe the present invention by way ofthe following non-limiting examples:

WORKING EXAMPLES General Methods Expression Vector

The nucleic acid fragment corresponding to SEQ ID NO: 1 or 2 wasinserted into the multiple cloning site of the pEMS1313 (see FIG. 1) toproduce the expression vectors used in the experiments.

Derivation of mEMS1202 Embryonic Stem Cells

Blastocysts were obtained from natural mating of B6-Hprt1^(b-m3)homozygous females to 129-ROSA26 heterozygous males at 3.5 dpc.Blastocysts were flushed from uterine horns as per Hogan et al. (1994),cultured in EmbryoMax® KSOM with ½ Amino Acids, Glucose and Phenol Red(Cat #MR-121, Millipore/Chermicon, Temecula, Calif.) for 3-5 h, and thentransferred onto mitomycin C (mitC; Cat#M4287, Sigma, Oakville, ON)mitotically inactivated B6-Hprt1^(b-m3), B6129F1, or 129 mouse embryonicfeeders (MEFs) derived from 13.5-day post-coital embryos (Ponchio et al.2000) in 96-well plates containing KSR-ESC (Knockout™ D-MEM,Cat#10829-018, Invitrogen, Burlington, ON) with 2 mM L-glutamine(Cat#25030-081, Invitrogen, Burlington, ON), 0.1 mM MEM nonessentialamino acid solution (Cat#11140-050, Invitrogen, Burlington, ON) and 16%Knockout™ Serum Replacement (Cat#10828-028, Invitrogen, Burlington, ON))media (MEF media was replaced 3-5 hour prior to transfer). Blastocystswere cultured as per (Cheng et al. 2004) with the followingmodifications: Cells were cultured for 7-9 days in KSR-ESC with minimaldisturbance (checked on day 2 to determine if the blastocysts had‘hatched’ out of the zona pellucida) and no media changes. Blastocystswhich hatched and had a well developed ICM (inner cell mass) weretreated with 20 μl 0.25% trypsin-EDTA (Invitrogen, Burlington, ON) for 5min at 37° C., triturated with a 200 μl pipetman, inactivated with 30 μl0.5 mg/ml soybean trypsin inhibitor (Invitrogen, Burlington, ON), andbrought up to 200 μl with KSR-ESC, then transferred individually to a24-well MEF plate containing 1800 μl KSR-ESC, for a total volume of 2ml. Beginning 4 days later, KSR-ESC media was replaced with FBS-ESCmedia (DMEM (Cat #11960-069, Invitrogen, Burlington, ON) with 2 mML-glutamine (Invitrogen, Burlington, ON), 0.1 mM MEM nonessential aminoacid solution (Invitrogen, Burlington, ON), 16% ES Cell Qualified fetalbovine serum (FBS, Invitrogen, Burlington, ON) and 0.01%β-mercaptoethanol (Sigma, Oakville, ON)) in 25%, 50%, 75% proportions(respectively) to adapt the cells to FBS containing media. On day 7 thecells were trypsinized to one well of a 24 well plate containing 1 ml of100% FBS-ESC media, with daily media replacement. Once confluent, wellscontaining ESC colonies were expanded 3×24 wells (with MEFs), thenpassaged to 3×24 (with MEFs) and 3×12 well (plastic—no MEFs) for DNAanalysis. Once confluent, the 3×24 wells were combined, aliquoted (3vials), and frozen in ESC-freeze media (50% FBS, 40% FBS-ESC media, 10%DMSO (Sigma, Oakville, ON), and the 3×12 well treated with lysis buffer(Fisher Scientific, Ottawa, ON), mixed and aliquoted. Cultures weregenotyped for X & Y chromosomes (Clapcote and Roder 2005),Gt(ROSA)26Sor^(tm1Sor) and WT alleles and Hprt1^(b-m3) and WT alleles.B6129F1-Gt(ROSA)26Sor^(tm1Sor)/+, Hprt1^(b-m3)/Y (mEMS1204 series) andB6129F1-Gt(ROSA)26Sor^(tm1Sor)+/+, Hprt1^(b-m3)/Y (mEMS1202 series) celllines were identified.

Knock-in at the Hprt1 Locus

The CLDN5 expression vector plasmid DNA was purified with Qiagen MaxiKit (Qiagen, Mississauga, ON), resuspended in 10:1 Tris-EDTA (TE, pH7.0)buffer, and linearized with I-Scel (New England Biolabs, Pickering, ON).Linearized plasmid DNA was resuspended in 85 μl of TE (10:0.1) to afinal concentration of 187.5 ng/μl, mEMS1202 ESCs were grown toconfluence on 4-6 T75 flasks of mitC treated Hprt1^(b-m3) mouseembryonic feeders (MEFs) in FBS-ESC media. ESCs (1.7-2.5×10⁷) in 720 μl1× PBS were added to the linearized DNA and electroporated in a 4 mmelectroporation cuvette (Bio-Rad Genepulser, Mississauga, ON), at 240 V,50 μF, 6-10 msec pulse, immediately resuspended in a total volume of 5ml of FBS-ESC media and plated onto 5×100 mm dishes of mitC B6129F1 MEFsin a total volume of 12 ml/100 mm dish. 24-36 h post-electroporation,correctly targeted homologous recombinants were selected for using HATmedia (FBS-ESC media containing 1× HAT ((0.1 mM sodium hypoxanthine, 0.4mM aminopterin, 0.16 mM thymidine), Cat#21060-017, Invitrogen,Burlington, ON). HAT media was changed every day for the first 3 days,and then every 3^(rd) day thereafter, for up to 10 days. Individualcolonies were counted and, typically, no more than 2 isolated colonieswere picked per 100 mm dish to optimize for independent homologousrecombination events. These colonies were expanded under standardprotocols for verification of the desired recombination event.

Derivation of Knock-In Mice

Chimeric mice from targeted ESCs were generated by microinjection (Hoganet al. 1994) into E3.5 blastocysts followed by implantation into theuterine horns of 2.5 day pseudopregnant ICR females. Chimeras wereidentified and coat color chimerism determined as outlined below.

Male chimeras derived from the mEMS1202 cell lines were mated withB6-Alb females, and germline transmission identified by the presence ofthe dominant Ty⁺ (tyrosinase; wild type) and the A^(W) (nonagouti; whitebellied agouti) or a (nonagouti; nonagouti) alleles making the progenyappear brown with a cream belly or black, respectively. Non-germlineprogeny were homozygous for the recessive Ty^(c-2J) (tyrosinase; albino2 Jackson) allele and appear white. All germline female offspring shouldcarry the knock-in X Chromosome and were mated with B6 males. N2offspring were analyzed for the presence of the KI allele by PCR.

Determination of Coat Color Chimerism

mEMS1202-derived chimeras were identified and level of coat colorchimerism determined as follows. mEMS1202 ESCs, heterozygous A^(w)/a andhomozygous for the wild type Tyr⁺ alleles will produce chimeras withagouti and black patches on a white background when injected into B6-Albblastocysts. The agouti patches result from melanocytes derived solelyfrom the ESCs (A^(w)/a, Tyr⁺/Tyr⁺), whereas ‘black’ patches result frommelanocytes that are a mixture of ESC (A^(w)/a, Tyr⁺/Tyr⁺) and host(a/a, Tyr^(c-2J)/Tyr^(c-2J)). For E14TG2a injections into B6 andmEMS1202 injections into B6-Alb, overall chimerism was calculated bysumming the percent of coat color patches derived solely from the ESC,plus half the percent of the ESC+ host areas, where we conservativelyestimated that half the melanocytes derive from the ESC and half fromthe host.

Reporter Gene Detection

Adult male chimeric and age matched control mice were perfused with 4%paraformaldehyde (PFA) as previously described (Young et al. 2002).Whole brains were dissected out and post-perfusion immersion fixed withPFA for 2 hours at 4° C. Brains were then transferred to 25% sucrose at4° C. overnight with gentle shaking. The brains were cryostat sectionedsagittally at 1 mm and sections were mounted in 12-well tissue cultureplates. LacZ expression was detected by using 5-bromo-4-chloro-3-indolylβ-d-galactopyranoside (x-Gal) as the substrate. The x-Gal stainingsolution contained the following chemicals: 1.0 mg/ml X-Gal, 2 mMpotassium ferricyanide, 2 mM, potassium ferrocyanide, and 40 mM MgCl₂ inPBS. In brief, slide mounted brain sections are rinsed with phosphatebuffered saline (PBS), then incubate with x-Gal (Boeringer Mannheim,Indianapolis, Ind.) at 37° C. from two hours to overnight. After theplates were taken out of the incubator they were rinsed with PBS andmoved into PBS for storage. Bright field images were visualized on aLeica MZ125 dissecting microscope and photographed with an OlympusCoolsnap cf color camera.

Example 1 Selection of CLDN5 Promoter Elements

Under the assumption that sequences under selective pressure will bemore conserved than those that are not, cross-species comparisons, orphylogenetic footprinting, were utilized to identify regulatory regions.The two mammalian species with a desirable evolutionary distance to usefor this approach are human and mouse. In the specific case of CLDN5,the conservation level between human and mouse was computed taking intoconsideration the non-coding sequence surrounding the CLDN5 gene. Forthis genomic region including a lot of non-coding sequences conserveddown to the frog, a threshold of 70% of identity was set up to selectcandidate regulatory regions. The CLDN5 basal promoter (SEQ ID NO: 6)and three upstream regulatory regions (SEQ ID NOs: 3, 4, 5) were chosenbased on these criteria.

Example 2 Expression of Reporter by CLDN5 Promoter Elements

The CLDN5 DNA expression vectors comprising the CLDN5 promoter elementcorresponding to SEQ ID NO: 1 (basal promoter only) and SEQ ID NO: 2(regulatory regions corresponding to SEQ ID NO: 3 and 4 fused to basalpromoter SEQ ID NO: 6) was introduced into mouse embryonic stem cells(ESCs) at the HPRT locus. The ESCs were used to generate geneticallymodified mice containing CLDN5 mini-promoters. Immunohistochemical andimmunofluorescence analysis of mouse brain tissue slices revealedexpression in the blood vessels throughout the brain and also in spinalcord. FIG. 2 shows expression from the basal promoter (SEQ ID NO: 1) inthe blood vessels of the brain. FIG. 3 shows expression from the basalpromoter with two upstream regulatory regions (SEQ ID NO: 2, comprisingSEQ ID NO: 3, 4 and 6 fused in non-native conformation) in the bloodvessels of the brain. FIG. 4 shows expression from this same constructin spinal cord. Expression was also observed in the heart, and weakexpression was observed in the liver and thymus. No expression wasdetected in lung.

Sequence Descriptions

-   SEQ ID NO: 1 , human CLDN5 mini-promoter element, basal promoter    element only;-   SEQ ID NO: 2 , human CLDN5 mini-promoter−basal promoter+regulatory    elements 1 and 2;-   SEQ ID NO: 3 , human CLDN5 regulatory element 1;-   SEQ ID NO: 4 , human CLDN5 regulatory element 2;-   SEQ ID NO: 5 , human CLDN5 regulatory element 3; and-   SEQ ID NO: 6 , human CLDN5 basal promoter element.

REFERENCES:

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All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. An isolated polynucleotide comprising a CLDN5regulatory element operably joined to a CLDN5 basal promoter through anon-native spacing between the promoter and the regulatory element. 2.The isolated polynucleotide of claim 1, operably linked to anexpressible sequence.
 3. An isolated polynucleotide comprising a CLDN5basal promoter.
 4. The isolated polynucleotide of claim 3, operablylinked to an expressible sequence.
 5. A vector comprising the isolatedpolynucleotide of claim
 1. 6. A vector comprising the isolatedpolynucleotide of claim
 3. 7. A cell comprising the vector of claim 5.8. The cell of claim 7, wherein the vector is stably integrated into thegenome of the cell.
 9. The cell of claim 8, wherein the cell is a bloodvessel cell or a stem cell.
 10. A cell comprising the vector of claim 6.11. The cell of claim 10, wherein the vector is stably integrated intothe genome of the cell.
 12. The cell of claim 11, wherein the cell is ablood vessel cell or a stem cell.
 13. A method of expressing a sequenceof interest, the method comprising: operably linking the sequence ofinterest to the polynucleotide comprising a CLDN5 regulatory elementoperably joined to a CLDN5 basal promoter through a non-native spacingbetween the promoter and the regulatory element; and introducing into acell permissive for expression from a CLDN5 promoter.
 14. A method ofexpressing a sequence of interest, the method comprising: operablylinking the sequence of interest to the polynucleotide comprising aCLDN5 basal promoter; and introducing into a cell permissive forexpression from a CLDN5 promoter.